The proposed project aims to understand the molecular underpinnings of inflammation-associated neoplasia. Patients with IBD continue to need colectomies for dysplasia largely because of our inability to halt IBD progression to cancer. Our previous work has demonstrated that innate immune signaling by toll-like receptor 4 (TLR4) contributes importantly to the development of colitis-associated cancer (CAC). We identified TLR4 as an oncogene which promotes colonic malignancy. We have shown that TLR4 is over-expressed in UC- associated dysplasia and in almost all CACs. We showed that animals deficient in TLR4 (TLR4-/- mice) are protected from colitis-associated neoplasia and have generated a mouse in which the villin promoter drives transgenic intestinal TLR4 overexpression (V-TLR4). These mice are highly prone to colitis-associated neoplasia. Mechanistically, we have shown that TLR4 promotes neoplasia by activating -catenin signaling through PI3 kinase in colonic epithelial cells. We present preliminary data that show that TLR4 drives expansion of Lgr5+ stem cells in both intestinal neoplasia and in normal mucosa suggesting that TLR4 signaling is driving stem cell self-renewal in this model in vivo. We also show that UC mucosa and TLR4- mediated inflammatory neoplasia in mouse models are characterized by an infiltrate of S100A8/A9 expressing myeloid-derived suppressor cells (MDSCs). In the current proposal we will test the hypothesis that TLR4 signaling plays both an autocrine role in malignant epithelial transformation and a paracrine role through activation of MDSCs to stimulate -catenin activation, epithelial cell proliferation and cancer stem cell expansion. This is pursued i the following specific aims: 1) Determine the role of TLR4 signaling in activation of intestinal stem cells in inflammatory neoplasia. Using our various TLR4 expressing animal models crossed to stem cell reporter Lgr5-EGFP mice we will investigate if TLR4 is required for expansion of lineage committed progenitors and of colon cancer stem cells during inflammation and neoplastic transformation in vivo. 2) Determine the role of TLR4 signaling in promoting stemness of colonic epithelial cells. We will use primary colonoid cultures to test if TLR4 signaling alters the phenotype (growth, budding) of primary colon stem cells in culture. We will use colon cancer cell lines to test whether TLR4 signaling promotes a cancer stem cell phenotype in vitro and increases tumorgenicity in vivo using xenografts. 3) Determine if S100A8/9+ MDSCs drive TLR4-dependent -catenin activation, stem cell activation, and tumorigenesis. We will determine whether S100A8/9 MDSCs are required for intestinal stem cell expansion and CAC. We will test if S100A8 or 9 activate -catenin in colonic epithelia in a TLR4-dependent manner. Human UC-associated MDSCs will be isolated and studied for their ability to stimulate -catenin signaling in colonic epithelia in vitro. The work proposed herein aims to provide the mechanistic justification for subsequent human studies to target innate immune signaling as a means to halt progression UC/inflammation to dysplasia.